NAD and NADH are the most abundant coenzymes included in the body, and participate in oxidation-reduction reactions of various dehydrogenases by means of reversible changes between NAD and NADH which give and receive hydrogen atoms. NAD and NADH have different absorption peaks at about 260 nm and 340 nm respectively, and changes in their absorbancy are utilized for measurement of activities of various enzymes.
A signal amplification system associated with the cyclic interconversion or cyclic oxidation-reduction reaction of NAD/NADH is known as a method for detection of NAD, or NADH in a high sensitivity (C. Bernofsky et al., Analytical Biochemistry, Vol. 52, pp. 452-458, 1973: A. Johannsson et al., Clinica Chimica Acta, Vol. 148, pp. 119-124, 1985: F. J. Dhahir et al., Clinical Chemistry, Vol. 38, pp. 227-232, 1992 and U.S. Pat. No. 4,769,321).
In this method, a color-development signal is amplified by repetition of the oxidation-reduction reactions between NAD and NADH (enzymatic cycling) to thereby detect the presence of NAD or NADH with a high sensitivity. Therefore, by correlating the presence or non-presence of NAD or NADH with a substance to be detected, the substance can be detected indirectly via the detection of NAD/NADH by this method.
As will be seen from the reaction diagram shown hereunder, NAD or NADH act as a coenzyme, in the presence of dehydrogenase (e.g., alcohol dehydrogenase which is abbreviated hereinafter as ADH) and its substrate (e.g., ethanol) for the oxidation reaction of the substrate, during which NAD is reduced to NADH. Furthermore, NADH is oxidated into NAD in the presence of a tetrazolium dye and its reductase (diaphorase), at the same time the tetrazolium dye is reduced to formazan as the color-development signal. Under the conditions mentioned above, the oxidation-reduction reaction between NAD and NADH is repeated to thereby gradually increase the signal intensity. ##STR1##
The detection technique of NAD or NADH with such a signal amplification system is applied to measurement of phosphatase activity (A. Johannsson et al., supra: and F. J. Dhahir et al., supra), and to measurement of NAD-synthetase activity (Hideo Misaki, Bio Industry, Vol. 7, pp. 775-787, 1990) and the like. In spite of the fact that the method is a colorimetric analysis, the method is accepted as an extremely excellent method in the field of immunoassay, especially in the measurement of alkaline phosphatase, since it can provide a potent detection sensitivity which is comparable to that in fluorescence technique (or fluorescent antibody technique) or color-development technique [Edited by Ishikawa et al., Kouso Men-eki Sokuteiho (in Japanese), the third edition, pp. 58-60, published by Igakushoin].
Heretofore, ADH derived from baker's yeast has been utilized in general in an analytical system utilizing such an enzymatic cycling (repetitive interconversion) of NAD/NADH. The ADH of baker's yeast, however, has a very short activity-duration so that a large quantity of ADH must be added in a reagent for the analytical system to enable the repetitive oxidation-reduction reaction between NAD and NADH which in turn amplifier a detectable color-development signal.
The present inventors have exerted their efforts to increase the sensitivity of the enzymatic analysis and stability of dehydrogenase activity acting as a catalyst to provide hydrogen atoms to the system of enzymatic cycling between NAD and NADH, and found that ADH derived from microorganisms belonging to Zymomonas has an extremely potent activity and that amino acid dehydrogenase (hereinafter abbreviate as AADH) derived from thermophilic microorganisms has an extremely stable activity compared with that of ADH derived from baker's yeast.